DESCRIPTION: Aspergillus fumigatus is the most frequent cause of invasive aspergillosis, an infection that has markedly increased in incidence over the past two decades. This is due, in part, to the growing population at risk for invasive disease. These patient populations are well defined, but little is known about the virulence factors of the organism and how they might influence the increasing incidence. Targeted approaches, taken to investigate the roles of proteinases and restrictocin, for example, have not proven very fruitful in identifying factors necessary for virulence. Basic knowledge of the pathogenic mechanisms employed by an organism is a prerequisite to identifying changes in these mechanisms that may contribute to increasing incidence of infection. Therefore, in this proposal, a novel approach to studying the virulence factors of A. fumigatus is proposed. By analogy with bacterial systems, it is presumed that during pathogenesis, numerous genes of A. fumigatus are activated and/or repressed. Identification of these regulated genes is a way to determine which genes or gene products are important in pathogenesis. During dissemination, the fungus invades blood vessels. Following hemotogenous spread, the vessel wall is breached as the fungus invades new tissue. This process involves the intimate interaction of the fungus with endothelial cells linig the vessels. Therefore, this application proposes to identify genes that are transcriptionally activated by A. fumigatus when grown on endothelial cells. The genes will be identified by differential display of mRNA; probes generated in differential display will be used to probe cDNA and genomic libraries of A. fumigatus. Gene disruptions will be made by interrupting an exon with the hygromycin resistance gene. Transformants that have ablated gene function via homologous integration will be tested for changes in virulence, both on the endothelial cell monolayers and in immunosuppressed mice. Preliminary data supports this as a viable conceptual approach. Over twenty genes have been identified to date. Two of these have been isolated from a cDNA library, and partial sequence analysis reveals that one is a putatively novel transcript that has been induced 9-fold and one is a member of the ras gene family.